Solvent-free process for obtaining phospholipids and neutral enriched krill oils

ABSTRACT

Organic solvent-free processes for obtaining krill oil compositions are disclosed. The processes include a) cooking krill in a cooker vessel for a time and at a temperature sufficient to denature the protein content of the krill and cause a first solid krill fraction and a first liquid krill fraction to be formed while substantially avoiding emulsification of the first solid and first liquid krill fractions; b) removing the first solid and first liquid krill fractions from the cooker vessel at a temperature of at least about 90° C.; c) separating the first solid fraction and the first liquid fraction; and d) obtaining krill oil with neutral enriched from the first liquid fraction, and e) by pressing of first solid fraction to obtain press liquid or a second liquid krill fraction for obtaining krill oil with phospholipids enriched krill oil, the separating and the obtaining steps being carried out without the use of organic solvents. Krill oil compositions made by the process are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation-in-Part of PCT/IB2009/007269 filed Oct. 30, 2009, the contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to processes for obtaining krill oil with poly-unsaturated fatty acid docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in the phospholipids fraction, with astaxanthin, and neutral lipids coming from the meal processing line, used for human health applications while the resulting krill meal will have a low fat content. This invention also discloses a procedure for obtaining a dried complex that contains krill oil in combination with phospholipids linked to DHA and EPA, proteins and astaxanthin for the use on human-health human applications.

BACKGROUND ART

Krill corresponds to a group of small and abundant marine crustaceans in the order Euphasiaceae, living in the pristine Antarctic Ocean that is considered the feed base of the all Antarctic eco-system. The Antarctic krill, in particular those that live at the Antarctic and sub-Antarctic regions, are composed of 11 Euphasia species, being dominant Euphausia superba, Dana and Euphausia. crystallorophias.

In recent years, krill has acquired great interest as a potential source of protein and other active biological products (Ellingsen, T. and Mohr, V. 1979. Process Bioch. 14:14; Suzuki, T. 1981. Fish and krill protein processing technology. London, Applied Science Publishers). The large expectation ciphered in the South Antarctic krill is based in the large biomass existent at the Antarctic oceans, estimated between 100 to 500 millions tons. It has been suggested that captures of krill could reach up to 50-100 millions tons/year, quantity that is equivalent to the total fish capture in the world (Budzinski, E., Bykowski, P. and Dutkiewicz, D. 1986. Posibilidades de elaboración y comercialización de productos preparados a partir de krill del Antartico, FAO Doc. T,c. Pesca (268):47p).

There are several publications related to the krill lipid content and composition (Grantham, G. J. 1977. The Southern Ocean. The utilization of Krill. Southern Ocean Fisheries Survey Programme, Rome. FAO GLO/SO/77/3:63p; Budzinski et al. 1986. Loc. cit.; Ellingsen and Mohr. 1979. Loc. cit.). The lipid content is about 10-26% of the krill dry weight, depending on the season of the year and its sexual maturity as well as body size. Usually the krill fatty period is between March to June each year. The female krill has near double amount of lipids than the male. The lipid concentration increases with age and decreases rapidly after the spawning. Krill lipids distribution studies showed that lipid rich areas are located along the digestive tract, between the muscle bundles and under the exoskeleton (Saether, O., Ellingsen, T. and Mohr, V. 1985. Comp. Biochem. Physiol. 81B:609).

The main krill lipid fractions include triglycerides, phospholipids, as well as its sterols and esters (Grantham. 1977. Loc. cit.; Budzinski et al. 1986. Loc. cit.). The average content of phospholipids is about 69% and triglycerides about 26%. The krill phospholipids fraction, rich in polyunsaturated fatty acids, particularly 20:5 and 22:6, correspond to approximately 50% of total phospholipids.

There are several publications describing Krill lipid composition. The following being the most relevant among them:

-   1. Gordeev et al. described that E. superba contains about 5% of its     natural weight of extractable lipids, more than half of which are in     the form of phospholipids—phosphatidylcholine (33-36% of the lipids     total), phosphatidylethanolamine (15-17%), lysophosphatidylcholine     (3-4%), others (2-3%)—while triacylglycerols predominate (32-35%)     among the phosphorus-free components. In the first two phospholipids     the dominating fatty acid residue is the arachidonic acid residue     (more than 40% of the acyl residues total) and the amount of     eicosapentaenoic acid residues (C20:5w3) are about 13 and 28%,     respectively. (Gordeev, K. Y. et al. 1990. Fatty acid composition of     the main phospholipids of the Antarctic krill Euphausia superb.     Chemistry of Natural Compounds. 26: 143-147).     -   Fricke et al. described that Euphausia superba Dana lipid         composition was phosphatidylcholine (33-36%),         phosphatidylethanolamine (5-6%), triacylglycerol (33-40%), free         fatty acids (8-16%) and sterols (1.4-1.7%). Wax esters and         sterol esters were present only in traces. More than 50 fatty         acids could be identified and the major ones being 14:0, 16:0,         16:1(n-7), 18:1(n-9), 18:1(n-7), 20:5(n-3) and 22:6(n-3).         Phytanic acid was found in a concentration of 3% of total fatty         acids. Short, medium-chain and hydroxy fatty acids (C=10) were         not detectable. The sterol fraction consisted of cholesterol,         desmosterol and 22-dehydrocholesterol. (Fricke, H. et al. 2006.         Lipid, sterol and fatty acid composition of Antarctic krill         (Euphausia superba Dana). Lipids, 19:821-827). -   2. Falk-Petersen et al described that lipids of Arctic and Antarctic     euphausiids show a seasonally-dependent high lipid content, and     neutral lipids, whether wax esters or triacylglycerols, are     primarily accumulated for reproduction. The Arctic Thysanoessa     inermis and the Antarctic Euphausia crystallorophias contain high     levels of wax esters and higher concentrations of 18:4(n-3) and     20:5(n-3) and a lower ratio of 18:1(n-9)/(n-7) fatty acids in their     neutral lipids than those in the Arctic Thysanoessa raschii and the     Antarctic Thysanoessa macrura and Euphausia superba. Large amounts     of phytol in the lipids of T. raschii and E. crystallorophias during     winter suggest the ingestion of decaying algae originated from     sedimenting material or in sea ice. Thysanoessa raschii, T. macrura,     and E. superba have a high ratio of 18:1(n-9)/(n-7) fatty acids,     indicating animal carnivory (Falk-Petersen, S. et al. 2000. Lipids,     trophic relationships, and biodiversity in Arctic and Antarctic     krill. Can. J. Fish. Aquat. Sci. 57: 178-191). -   3. Clarke described lipid content and composition of the Antarctic     krill Euphausia superba. Female total lipid content increases during     the summer as the ovary matures, and there is also some evidence of     an increase in the lipid content of males and immatures as winter     approaches. The storage lipid is mainly triacylglycerol and there is     less than 1% wax ester. Fatty acids are moderately unsaturated,     though less so in the ovarian lipid, and the triacylglycerol     contains up to 4% phytanic acid (Clarke A. 1984. Lipid content and     composition of Antarctic krill, Euphausia superba Dana. J. Crust.     Biol. 4:285-294). Phleger et al. described lipid compositions of     Antarctic euphausiids, Euphausia superba , E. tricantha, E. frigida     and Thysanoessa macrura collected near Elephant Island during 1997     and 1998. Total lipid was highest in E. superba small juveniles (16     mg g-1 wet mass), ranging from 12 to 15 mg in other euphausiids.     Polar lipid (56-81% of total lipid) and triacylglycerol (12-38%)     were the major lipids with wax esters (6%) only present in E.     tricantha. Cholesterol was the major sterol (80-100% of total     sterols) with desmosterol second in abundance (1-18%). 1997 T.     macrura and E. superba contained a more diverse sterol profile,     including 24-nordehydrocholesterol (0.1-1.7%),     trans-dehydrocholesterol (1.1-1.5%), brassicasterol (0.5-1.7%),     24-methylenecholesterol (0.1-0.4%) and two stanols (0.1-0.2%).     Monounsaturated fatty acids included primarily 18:1(n-9)c (7-21%),     18:1(n-7)c (3-13%) and 16:1(n-7)c (2-7%). The main saturated fatty     acids in krill were 16:0 (18-29%), 14:0 (2-15%) and 18:0 (1-13%).     Highest eicosapentaenoic acid [EPA, 20:5(n-3)] and docosahexaenoic     acid [DHA, 22:6(n-3)] occurred in E. superba (EPA, 15-21%; DHA,     9-14%), and were less abundant in other krill. Lower levels of     18:4(n-3) in E. tricantha, E. frigida and T. macrura (0.4-0.7% of     total fatty acids) are more consistent with a carnivorous or     omnivorous diet as compared with herbivorous E. superba (3.7-9.4%).     The polyunsaturated fatty acid (PUFA) 18:5(n-3) and the very-long     chain (VLC-PUFA), C(26) and C(28) PUFA, were not present in 1997     samples, but were detected at low levels in most 1998 euphausiids     (Phleger, C. F. 2002. Interannual and between species comparison of     the lipids, fatty acids and sterols of Antarctic krill from the US     AMLR Elephant Island survey area. Comp Biochem Physiol B Biochem Mol     Biol. 131:733-747). -   4. Kolakowska described lipid compositions of seven krill (Euphausia     superba D.) samples, fresh and after various periods of storage at     251 K (Kelvin Degrees). Fresh krill lipid composition differed from     that determined in frozen samples, depending on storage duration,     season of harvest, and developmental stage. Phospholipids proved     most susceptible to changes, as opposed to triglycerides, which were     most resistant; diglycerides and cholesterol esters were also     destroyed. The freezing process per-se affected the lipid     composition only slightly; however, after 30 days storage the amount     of free fatty acids almost doubled. After 6 months storage at 251°     K, 70% of phospholipids were decomposed and the amount of free fatty     acids increased by a factor of 6 to 20. Monoglycerides, absent from     fresh krill, appeared after several months of frozen storage.     Juvenile krill were more susceptible to lipolytic changes. Females     bearing mature eggs contained stable phospholipids; only     triglycerides were hydrolysed (Kolakowska A. 1986. Lipid composition     of fresh and frozen-stored krill. Z Lebensm Unters Forsch.     182:475-478. Bottino described lipid compositions of two Antarctic     euphausiids. In Euphausia superba complex lipids were the major     lipid class followed by triglycerides. In E. crystallorophias the     complex lipids were also the major lipid class, but the second major     constituent was wax. The complex lipids of both Euphausiids     consisted mostly of phosphatidylcholine with smaller amounts of     phosphatidylethanolamine and lysophosphatidylcholine. The     phospholipids of E. crystallorophias were less unsaturated than     those of E. superba. The waxes of E. crystallorophias were mostly     esters of oleic (84%) and palmitoleic (10%) acids with     n-tetradecanol (69%) and n-hexadecanol (28%) (Bottino, N. R. 1975.     Lipid composition of two species of Antarctic krill: Euphausia     superba and E. crystallorophias. Comp Biochem Physiol 13.     50:479-484). -   5. EP1997498 and WO02/102394 owned by Neptune Technologies &     Bioressources Inc. Relate to Neptune krill oil that corresponds to     acetone extracted krill lipids. Proteins and krill material are     removed from the lipid extract through filtration. The acetone and     residual water are removed by evaporation. The phospholipids content     is 38-50%, and EPA and DHA is 22-35%, wherein the major amount of     these omega-3's are attached to phospholipids. -   6. US2008/0274203 and WO 2008/117062 owned by Aker Biomarine ASA.     These applications disclose a new krill oil composition     characterized by high amounts of phospholipids, astaxanthin esters     and/or omega-3. This krill oil is characterized by comprising about     30-60% w/w phospholipids and about 20-35% omega-3 fatty acid wherein     the major amount of these omega-3 lipids are attached to     phospholipids.

The high content of polyunsaturated fatty acids in the phospholipidic fraction could be necessary to keep the plasmatic membrane fluidity at low temperatures in the Antarctic oceans. A high unsaturation level might be required to give the krill phospholipids deposits the necessary plasticity for the animal flectation and motion at low temperatures.

An increase of the total lipids present in the krill is accompanied with both a decrease in the phospholipids and an increase in the triglycerides. The polyunsaturated fatty acid content decreases as the content of total lipids increase (Saether et al. 1985. Loc. cit.). Post-mortem changes that occur in krill lipids showed that during the krill storage at 0° C. the polyunsaturated fatty acids (20:5, 22:6) compared to the content of fatty acids (16:0) do not decrease. These data suggest that during the krill storage at 0° C. a large oxidation of polyunsaturated fatty acids after the crustacean death does not occur.

There are several documents describing industrial processes to obtain krill oil. These documents include:

-   1. Budzinski et al. (1986. Loc. cit.) and Saether et al. (1985. Loc.     cit.), described procedures for krill lipid extraction with     different organic solvents. -   2. CA2346979, ES2306527 or UA75029, documents presented by     Universite de Sherbrooke. This document described a method to     extract lipid fraction from marine and aquatic animals, including     krill, using acetone extraction. -   3. WO2006/106325 presented by Pro-Bio Group AS. This document     described a process to obtain phospholipids from krill. This process     comprises contacting the krill meal with an organic solvent to     produce a lipid containing liquid. Optionally this liquid is     extracted with other organic solvents to extract neutral lipids. The     remaining fraction is a phospholipids enriched fraction. -   4. EP1997498 and WO02/102394 presented by Neptune Technologies &     Bioressources These documents describe lipid extraction from krill     or krill derived material utilizing ketone solvents, preferably     acetone. -   5. GB407729 presented by Johan Olsen Nygaard. This patent describes     a method to extract oil from marine animals, in particular whale and     other sea mammals, by heating in bath of oil. This document is     different from our invention because is not applied to krill and     utilizes heated oil for lipid extraction. Our invention does not use     heated oil for phospholipids extraction. -   6. WO2009/027692 discloses a multiple step process for obtaining     krill meal and related products. The process includes a first     heating step in which krill is brought to a temperature of about     75° C. and thereafter sieved to obtain phospholipid free krill. This     phospholipid free krill is then heated a second time to 85° C.,     sieved again and pressed. The krill liquid (milk) left behind from     the first heating step is then coagulated to remove the proteins and     phospholipids.

These five documents describe the utilization of organic solvents, particularly acetone, to extract krill lipid fractions. These methods being different from the procedure disclosed herein, as the method declared in the present invention does not utilize organic solvents for extracting or purifying lipid fractions from Antarctic krill.

-   1. JP58008037, Nippon Suisan Kaisha Ltd. This document describes a     method to obtain eicosapentaenoic acid or derivatives from Antarctic     krill oil. Krill oil is converted into free fatty acid or an ester     thereof by the conventional method, e.g. saponification or     alcoholysis, being the resultant product continuously distilled to     collect a main fraction of distillate containing 40 wt % or more     titled substance. The main fraction of distillate is then treated     with urea to remove low unsaturated fatty acids. This document     differs from the present invention through the use of a distillation     process for lipid purification. -   2. JP2004024060 (Nippon Suisan Kaisha Ltd). This document describes     a procedure for obtaining an astaxanthin enriched lipid fraction     from Antarctic krill. This document declares a different process     from the one declared in the present invention in that the instant     process aim is not the astaxanthin production. -   3. US2008/0274203 (Aker Biomarine ASA). This document describes a     procedure for obtaining krill oil from krill meal using     supercritical fluid extraction in a two-stage process. Stage 1     removes the neutral lipid by extracting with neat supercritical CO₂     or CO₂ plus approximately 5% of a co-solvent. Stage 2 extracts the     actual krill oils by using supercritical CO₂ in combination with     approximately 20% ethanol. This document differs from the present     invention as this procedure does not use supercritical fluid     extraction. WO2007/080514 (Krill A/S and Alfa Laval Copenhagen A/S),     describes a method for extracting lipid fractions from krill,     wherein freshly captured krill is grinded to obtain a slurry, which     is gently heated to a temperature below 60° C., preferably bellow     30° C., for less than 45 minutes, thereafter the liquid splits into     an aqueous phase and a krill oil phase from which a krill oil     extract is derived without the use of organic solvents. This     document reveals a process different from the one declared in the     instant invention as the present procedure does not grind the     captured krill before heating. This document is also different since     the heating temperature declared in the instant process is >90° C.     Another main difference regarding the process declared in this     document is that grinded krill used to produce slurry before heating     produces an emulsification that impedes the phospholipids separation     by centrifugation. In the present invention a slurry is not directly     or indirectly produced. Further in WO2007/080514 is used ultrasound     for krill oil separation while the present invention does not use     any type of ultrasound technology. It also proposes a simple     extraction while the present invention is based in a     double-extraction principle. -   4. WO2007080515 (Aker Biomarine ASA) describes a process for     obtaining krill lipids by processing the krill at a temperature     below 60° C. with mechanical and physical disruption of the lipid     cell membrane to facilitate low temperature extraction. This process     takes place under inert gas atmosphere to prevent oxidation or     denaturation of fat and proteins. Intermediate processing tanks are     kept at a minimum level to reduce residence tithe; and the oil after     recovery is immediately frozen to stabilize it. This document     differs from the present invention since the instant procedure does     not grind the captured krill to facilitate lipid extraction, instead     a heating temperature of >90° C. is used; further the process of the     present invention does not use any type of gases or freezing     technologies. -   5. WO2008/060163 (Pronova Biopharma Norge AS), describes a procedure     for obtaining krill oil-using CO₂ at supercritical pressure     containing ethanol, methanol, propanol or isopropanol. This document     is different from the present invention because the present     procedure does not use CO₂ supercritical lipid extraction with or     without solvent. -   6. WO2009/027692 (Aker Biomarine ASA), describes a method for krill     meal production.

This procedure uses a two-step cooking process. In the first step the proteins and phospholipids are removed from the krill and precipitated as a coagulum. In the second stage the krill without phospholipids are cooked. Following this, residual fat and astaxanthin are removed from the krill using mechanical separation methods. In this method, krill is heated to 60-75° C. in the presence of water to dissolve/disperse lipids and proteins to the water phase, called krill milk. This krill milk was heated to 95-100° C. to remove as a precipitate the krill protein and lipids from the water phase. The processes of the present invention differ from those disclosed in this document since the krill oil is not separated from the crustacean by precipitation and the multiple heating steps are avoided.

From current traditional krill meal processing on board, in some factory vessels, only a small amount of krill oil is produced. This krill oil is usually enriched in neutral lipids with very low or undetectable amount of phospholipids (<0.5%). Normally, during the traditional on board krill process, fresh krill is heated using an indirect heating cooker with rotating screw conveyor, followed by a twin-screw press and drier. The press liquid obtained by the twin-screw press is passed through a decanter to remove the insoluble solids. The clarified decanter liquid is then used to feed separators centrifuges to separate the krill oil normally enriched with neutral lipids and astaxanthin. In this traditional process the phospholipids are bound to the proteins in the press cake. Therefore, phospholipids are usually found associated to the krill meal. In the krill fatty period, the fat content in the krill meal is usually 16-18%. In the same krill fatty period, the yield of the neutral lipid-enriched krill oil obtained using the traditional krill meal plant is low, ranging usually between 0.3-1.0% from raw krill. This neutral lipid-enriched krill oil contains astaxanthin ranging between 700-1.500 mg/kg depending on the season and the fishing ground catching.

Moreover, when a non-traditional krill meal processing layout is used, a similar situation explained above was obtained. Normally, the non-traditional krill meal plant considered a contherm cooker system, a two-phase decanter or three-phase decanter and a drier. These decanters are used for de-watering and de-fatting the cooked krill. The decanter liquid is used to feed the centrifuge separators to obtain usually a neutral lipid-enriched krill oil with low or undetectable levels of phospholipids (<0.5%). In this case, the phospholipids are also bound to the proteins in the decanter solids. As described above, phospholipids are found in the krill meal.

In this case, the yield of neutral lipid-enriched krill oil from the non-traditional krill meal plant is much lower, in the fatty period ranging from 0.1 to 0.4% of raw krill. In this process a conventional contherm cooker system is used which has inherent agitation (scraped knife). Therefore, the processed krill is agitated and also minced. This agitation/mincing process produce lipid emulsification along with krill proteins and water. Besides, krill phospholipids catalyze the emulsification because these lipids act as an emulsifier agent. For this reason, using a non-traditional krill meal process, higher lipid content is bound to the decanter solids. In the krill fatty period, the fat content of the decanter liquid has a lipid content ranging from 1.0 to 2.5%, the resultant stickwater has a yellow color and it is emulsified, with a fat content ranging from 0.7 to 1.6%, the krill meal has a fat content ranging from 20 to 26%, and the krill oil recovery by this non-traditional process is very low.

The lipid composition and fatty acid profile of neutral lipid-enriched krill oil obtained using the traditional and non-traditional krill meal processing are very similar: triglycerides about 86-89%, phospholipids not detected (<0.5%), and DHA and EPA about 4-6%.

Several efforts have been made to produce a krill meal with a lower fat content and phospholipids enriched krill oil containing DHA and EPA, through an industrial scale method associated to a krill meal plant. Several different cooking temperatures, different decanting torque, strong pressing, using two decanting steps, washing the first decanter solids with stick water before the second decanter, electroplasmolysis and so on have been tested. However, the results have not been successful.

Focusing on the problem to separate phospholipids-enriched krill oil with DHA and EPA, some extraction methods have been developed and patent protected.

The patent application CL 1021 1995 (Compañía Tepual S.A) refers to a method for obtaining krill oil using thermal fractionation and centrifugation. This oil obtaining process claims that the fatty acids types and lipids composition can be regulated controlling the krill cooking temperature during the process of oil production. At high cooking temperature (95° C.) lower yield of poly-unsaturated and phospholipids fractions were obtained as compared when the cooking temperature was reduced (75° C.). Also this oil process claims that main components of its fractional composition correspond to triglycerides being 35 to 96%; and phospholipids from 4 to 28%. The poly-unsaturated fatty acids ranged from 4 to 46%. Basically this process used for obtaining krill oil, considers krill cooking, pressing the cooked krill and the passing the press liquid through a two-phase decanter to separate the insoluble solids, being the oil separated from the decanter liquid using centrifuge separator. During this process only one type of krill oil was obtained.

SUMMARY OF THE INVENTION

This invention discloses a new on board (at sea) and/or on shore (on land) process for simultaneously obtaining both: 1) phospholipids enriched krill oil and 2) neutral lipid enriched krill oil containing DHA and EPA poly-unsaturated fatty acids and astaxanthin. Antarctic krill oil is particularly rich in DHA, and EPA containing phospholipids. Because the amphipathic nature of phospholipids, i.e. a negative charged phosphate group in one end and a hydrophobic lipid in the other end, this lipid is a very potent emulsifier agent. The grinding and/or mincing of krill induces emulsification of soluble proteins, water, neutral lipids and phospholipids. Therefore, once the emulsion is formed, phospholipids can be obtained from krill mixtures only by organic solvent or CO₂ supercritical extractions. See WO02/102394 and US200810274203 as example. The present invention advantageously avoids the use of these extractors and, as mentioned below, the grinding and/or mincing of the krill during the process.

In the present invention, a new procedure for phospholipids extraction from fresh krill or other similar crustaceans is disclosed. This procedure is based on a process where no grinding, agitation and/or mincing of the krill is carried out. Using this method, no or substantially no phospholipid emulsification occurs. Cooking of whole intact krill or fractions produces a first solid fraction and a first liquid fraction, with the first liquid fraction being released from the crustaceans. The oil contained in this liquid phase (i.e. the first liquid fraction) is enriched with neutral lipids. The first solid fraction obtained from cooking can be further processed to obtain a second liquid fraction and a second solid fraction. The oil obtained from this second liquid fraction is also enriched with phospholipids. The second solid fraction is processed to obtain a low fat krill meal.

The present invention allows a much more efficient extraction process and is preferably done almost immediately after raw or fraction of krill has been harvested at sea. The present invention is preferably done onboard, which allows having a much more controlled extraction process, allowing a dramatic reduction in not-necessary logistic transshipment, raw material and cargo handling. Other advantages of the present invention include the “finished product” character of the end product which is obtained on the same premises where capture and processing takes place.

The process of the present invention can also be used with other similar crustacean species, whether they are farmed or captured crustaceans, such as but not limited to Pandalus borealis, Cervimunida johni, Heterocarpus reedi, Pleuroncodes monodom, Penaeus vannamei, Penaeus monodon, Penaeus stylirostris, Penaeus chinensis, Penaeus. orientalis, Penaeus japonicus, Penaeus indicus, Penaeus merguiensis, Penaeus esculentus, Penaeus setiferus, Macrobrachium spp, and others. Moreover, the processes described herein can be used to obtain oil, extracts and useful compositions from various fish and/or algae species. In such embodiments, the processes are carried out but the krill is replaced with either fish or algae.

In accordance with one aspect of the invention, a solvent-free process for producing krill oil is provided which includes:

-   a) cooking krill, which is preferably fresh, for a time and at a     temperature sufficient to transform the krill into a form capable of     being separated into a first solid fraction and a first liquid     fraction while substantially avoiding emulsification of the krill; -   b) separating the first solid fraction and the first liquid fraction     from the cooked krill; and -   c) obtaining krill oil from either the first liquid fraction or the     first solid fraction, said separating and said obtaining steps being     carried out without the use of solvents.

In a related aspect of the invention there is provided a solvent-free process for producing krill oil which includes:

-   a) cooking krill, which is preferably fresh, in a vessel for a time     sufficient to cause the krill reach a temperature of at least about     90° C. and transforming the krill into a form capable of being     separated into a first solid fraction and a first liquid fraction     while substantially avoiding emulsification of the krill; -   b) removing the cooked, transformed krill from the vessel at said     temperature of at least 90 degrees; -   c) separating the first solid fraction and the first liquid fraction     from the cooked, transformed krill; and -   d) obtaining the krill oil from either the first liquid fraction or     the first solid fraction, said process being carried out without the     use of solvents.

To obtain the krill oil from the first liquid fraction, the first liquid fraction is separated into krill oil and water.

As noted above, the first solid fraction which results from cooking is, preferably, further processed by:

-   -   (a) squeezing the first solid fraction to obtain a second solid         fraction and a second liquid fraction; and     -   (b) separating the second liquid fraction into a krill oil and         water.

Preferably, the first liquid fraction contains a krill oil enriched with neutral lipids and the second liquid fraction krill oil is preferably enriched with phospholipids

Separating the second liquid fraction into krill oil and water is suitably accomplished by using a separator to separate the oil from the water, or by drying to remove the water from the oil.

As a part of the present innovation, this invention is carried out with equipment that avoids or minimizes the phospholipids forming emulsions while separating the phospholipids from the krill. The equipment is chosen from conventional equipment and is operated to avoid agitation, mincing, and/or grinding, throughout all processing steps (e.g. cooking, pumping, and so on) to reduce oil emulsification with protein, water and krill phospholipids, this last component acting as the emulsifier agent. Such equipment include screw pumps, continuous cookers with screw conveyor at low speed, belt conveyors, screw conveyors, de-boner, chopper or similar.

Other equipment possible to use, that avoids agitation and/or grinding, throughout all processing steps include:

-   1. Krill and other crustaceans peeling machines -   2. Krill and other crustaceans brush peeling machines -   3. Krill and other crustaceans disk peeling machines -   4. Krill and other crustaceans coagulator machines working at low     speed -   5. Krill and other crustaceans dewatering machines working at low     speed -   6. Krill and other crustaceans deboning machines.

This invention relates to a method for obtaining specific Krill oils from Krill, preferably from South Antarctic Krill, through a processing preferably made on board (at sea), on board factory trawlers, or mother vessels. Further the present invention is suitable for being carried out on shore (on land) processing plants. The final processing target of the processing method disclosed in this invention includes: a) cooking of whole or fraction fresh marine species without agitation and/or mincing; b) separating of cooked marine species using, for example, a two-phase decanter and/or a three-phase decanter or any other type of separator to obtain a partial de-fatted and de-watered solid, a first solid fraction, and a decanter liquid, a first liquid fraction,) c) squeezing of the partial de-fatted and de-watered decanter solid through a press and/or using a twin-screw press and/or any other pressing device to obtain a press liquid, a second liquid fraction, and a solid fraction, a second solid fraction, which can be further processed to obtain the low fat dried marine-species meal; d) centrifuging of the press liquid to obtain the phospholipids enriched marine-species oil; e) separating of the decanter liquid obtained in step b) to obtain the neutral lipid enriched marine-species oil and stick water. The equipment used in this process avoids agitation throughout all steps (i.e. cooking, pumping, and so on) to reduce oil emulsification with protein, water and marine-species phospholipids, acting this last component as the emulsifier agent.

This invention also includes a phospholipids-, neutral lipids-, omega-3's- and protein-enriched dried powder, and the use of this product as a heath product for human application, a powerful health-promoter, growth enhancer, immune-system promoter and wellness end-product. All products obtained with the procedure disclosed in this invention are produced by a non-chemical treatment, i.e. a process free of organic solvents and/or CO₂ supercritical fluid, for separating the krill oil.

The phospholipids enriched krill oil can be broadly defined as including:

-   -   (a) phospholipids in an amount of about 30 to about 70% by         weight;     -   (b) DHL and EPA in an amount of about 10 to about 70% by weight;     -   (c) neutral lipids in an amount of about 30 to about 70% by         weight;     -   (d) astaxanthin in an amount of about 200 to about 1,500 mg/kg;     -   (e) phosphatidylserine in amount of about 1.5% by weight or more         total lipids;     -   (f) lysophosphatidylcholine in an amount of about 0.6% or less         by weight total lipids; and     -   (g) free fatty acids in an amount of about 3.4% or less by         weight total lipids.

The Phosphatidylserine (PS) content of the PL (phospholipid)-enriched krill oil obtained as a result of the present invention, about 1.5% or more, is believed to be unique as compared to that obtained using prior art processes, i.e. those using solvents and/or CO₂ supercritical to extract PL-enriched krill oil. In those prior art processes, PS content is either nil or less than 0.5%.

The phospholipids enriched krill oil of the present invention is useful for human health applications and has a total phospholipids content of about 30 to 70% w/w, preferably from about 35 to 60% w/w, and more preferably from about 35 to 55% w/w. The DHA and EPA content are, in combination, from about 10 to 70% w/w, preferably from about 15 to 60% w/w, and more preferably from 20 to 55% w/w. Neutral lipids content is from 30 to 70% w/w, preferably from about 40% to 65% w/w, and more preferably from about 45 to 65% w/w. Astaxanthin content is from about 200 to 1,500 mg/kg, preferably from about 300 to 1,200 mg/kg, and more preferably from about 400 to 1,000 mg/kg.

The neutral lipid enriched krill oil can be broadly defined as including:

-   -   (a) neutral lipids in an amount of about 50 to about 100% by         weight;     -   (b) DHA and EPA in an amount of about 2 to about 45% by weight;     -   (c) phospholipids in an amount of less than about 10% by weight;     -   (d) astaxanthin in an amount of about 200 to about 1,500 mg/kg;         and     -   (e) free fatty acid content in an amount of about 3.4% or less         by weight.

The neutral lipid enriched krill oil of the present invention is also useful for human health applications, having a content of neutral lipids from 50 to 100% w/w, preferably from 60 to 100% w/w, and more preferably from 70 to 100% w/w. DHA and FPA content are from 2 to 45% w/w, preferably from 2 to 40% w/w, and more preferably from 5 to 35% w/w. Phospholipids content is less than 10% w/w, preferably less than 5% w/w, and more preferably less than 2% w/w. Astaxanthin content is from 200 to 1,500 mg/kg, preferably from 300 to 1,200 mg/kg, and more preferably from 400 to 1,000 mg/kg.

The phospholipids-enriched krill oil of the present invention is preferably produced on board (at sea) which provides having the direct advantage of using of a fresh raw krill, this way avoiding freezing of the whole or fraction raw of krill. In addition to the freshness of the said end material, on board processing also produces fresh krill meal, which can eventually be used for further krill oils extraction processes in on shore (on and) processing plants. In alternative aspects of the invention, the process can be carried out on land using fresh or frozen krill previously obtained from the sea, which would be preferably be at least partially thawed before being cooked, or krill portions previously processed in some manner.

These preferably on board (at sea) krill oil processing methods have additional and important advantages such as avoiding frozen raw krill transportation from at sea operations (on board) to on shore (on land) port facilities and further transportation to on shore (on land) processing plants, this way the end-products of present invention have a significantly lower cost structure.

The phospholipids-enriched krill oil of the present invention, which is preferably produced using on board (at sea) processes. That means the assurance of the use of highly fresh whole or fraction raw krill, avoiding phospholipids decomposition and/or lipids deterioration which may occur during certain freezing process, frozen transportation and frozen storage typically required when prior art processes are carried-out on land (on shore) premises.

This invention represents a potent krill-related environment-driven development, as among its direct benefits, it includes:

-   a) An overall lower catch-volume requirement to secure the same     amount output of end product as compared to other methods as there     is a more efficient processing which allows less captured-tonnage     per end-product-tonnage; -   b) a significant reduction in processing infrastructure in relation     to any other comparable processing method as the amount of catch and     equivalent processing capacity is diminished due to the onboard     processing; -   c) a relevant improvement of the end-product quality in terms of     freshness and its molecules quality, which means that the benefit to     the end-user, be it for human health or animal nutrition, will be     through the reduction of the market-cost per equivalent high-quality     molecule; -   d) this invention guarantees lower processing costs which goes to     the direct benefit of end-users through a lower end-product selling     price; -   e) the biomass protection is also guaranteed as the value-added     concept is secured on board (at sea) and does not need extra costs     to transport raw material for further on shore (on land) processing; -   f) this invention allows having end-products secured on board (at     sea); -   g) This invention will also provide a much better resource     protection as the catch effort will not be focused on a limited     catch and processing period rather spread-out the entire krill     fishing season.

This invention further relates to South Antarctic Krill processing made preferably on board (at sea) factory trawlers or mother vessels. This invention also relates to krill processing made on shore (on land) processing plants whereas the on board (at sea) processing steps prevails on shore (on land). The final processing target of the processing method disclosed in this invention includes:

-   a) cooking of whole and fresh marine species without agitation and     or grinding; -   b) decanting of cooked marine species, i.e. krill using, for     example, a two-phase decanter and/or a three-phase decanter and/or     any other type of solid-liquid separator to obtain a partial     de-fatted and de-watered solid and a decanter liquid; -   c) squeezing of the partial de-fatted and de-watered decanter solid     through a press, such as, for example, a twin-screw press to obtain     a press liquid and a solid fraction which is further processed to     obtain the low fat dried marine-species meal; -   d) centrifuging of the press liquid to obtain the phospholipids     enriched marine-species oil; and -   e) centrifuging of the decanter liquid obtained in step b) to obtain     the neutral lipid enriched marine-species oil and stick water, i.e.     the water remaining after the oil is removed from the aqueous phase.     Stick water contains a substantial percent of the total protein as     well as other soluble components of the krill.

This process uses equipment that avoid or substantially avoid agitation throughout all steps (i.e. cooking, pumping, and so on) to reduce oil emulsification with protein, water and marine-species phospholipids, which act as the emulsifier agent.

The on board (at sea) processing includes either on board factory trawlers, mother vessels acting as processors only, mother vessels acting as catchers and processors or any other combination for mother vessels, onboard tramper vessels used as processing factories and/or any other at sea processing factory vessel and/or any other at sea processing factory layout operation for this specific purposes, being the resulting marine-oils obtained either as a by-product or as a final product or a combination of both.

The krill meal obtained with the process of the present invention has a fat content ranging from 5 to 15%, protein content from 60% to 70%, and moisture content from 6 to 10%.

Because both krill oil compositions obtained in the present invention are not exposed to the use of any toxic or non-toxic chemicals during the process, the resulting krill oils of this invention could be safely used for human consumption according to the European Food Safety Authority (EFSA) (Scientific Opinion of the Panel on Dietetic Products Nutrition and Allergies on a request from the European Commission of the safety of “Lipid extract from Euphausia superba” as food ingredient. The EFSA Journal (2009). 938, 1-16). Human health applications for the krill oils obtained in the present invention, can be without limitation for reducing premenstrual symptoms, preventing hypertension, control of blood glucose levels in patients, control of arthritis symptoms, prevention of hyperlipidemia and other health applications. Such oils can also be used in preparing dermatologic products, especially topic or systemic products, for treating skin diseases related to a deficiency of essential fatty acids, such as xerotic skin, hyperkeratosis, ictiosis, acne, dermic ulcers, psoriasis, serborreic eczema, atopic dermatitis, among others.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 and 2 are flowcharts depicting the process of the present invention;

FIG. 3 shows a flowchart for a process to obtain neutral lipids rich krill oil in accordance with the invention; and

FIG. 4 shows a flowchart for a contherm process to obtain krill oil reviewed in Table 2 below.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it should be understood that the invention as described is not to be limited in its application to the process details described herein. The invention as such, embraces other embodiments and various ways for being applied. It is also should be understood that the phraseology or terminology used herein is for the purpose of description and it is not intended for any type of limitation.

The following description may be more readily understood by reference to FIGS. 1 and 2. The present invention provides systems and processes for processing a marine biomass. As a preferred embodiment, the marine biomass is krill, preferably the Antarctic krill Euphasia superba. Other krill species or crustacean can also be processed using the systems and processes of the present invention. Examples of such species are E. crystallorophias, E. frigida, E. tricantha, E. vellantini, E. lougirostris, E. lucens, E. similis, E. spinifera, E. recurva, E. pacifiea, Thysanoessa macrura, T. vicinia, T. gregaria, T raschii, T. inermis, Pandalus borealis, Cervimunida johni, Heterocarpus reedi or Pleuroncodes monodom. The krill is preferably processed in a fresh state as defined herein. Moreover, the processes described herein can be used to obtain oil, extracts and other useful compositions from various fish and/or algae species. In accordance therewith, any of the processes described herein is modified to replace the krill with the desired fish and/or algae species.

As a preferred embodiment, the krill is harvested 10, see FIG. 1, at sea in a conventional manner using conventional equipment and then processed fresh on board a fishing vessel either factory trawler, mother factory vessels, or intermediate processor or similar or whatever other ship suitable to carry out the process of this invention within a term of 14, 12, 10, 8, 6, 5, 4, 3, or preferably 2 or 4 hours of catching krill. In some embodiments, the krill is processed on board a ship within 1 hour or preferably 0.5 hours or more preferably within 20 minutes after catching the krill. Within the embodiments of the present invention, it is included that the ship tows a trawl that is configured to catch the krill and/or that the ship receives its krill or other species from fishing vessels or other factory trawlers. The krill is then transferred from the trawl to the ship and processed, preferably immediately after krill catching. The trawl comprises (either a regular fishing gear composed of trawls and/or purse seining system and/or) a pumping system to pump the freshly caught krill from the trawl to the ship so the krill can be processed in a fresh state. In some embodiments the catch system, be it regular trawl or pump system, it is designed in such a manner that substantially no damage, (deterioration) grinding or mincing is done to the krill during the pumping from the trawl to the ship.

In a preferred embodiment, the fresh whole krill or fraction is transported to the cooker or cooker vessel 12 using belt conveyor, or screw conveyor or screw pumping or another transport system avoiding krill agitation to avoid the oil emulsification with protein and water. Sea water drains from the krill and krill is preferably washed with fresh water prior to feeding the cooker. The whole krill is cooked, i.e. heated, in a cooker vessel with indirect and/or direct steam heating or another heating system. Combinations of indirect heating and live steam injection can also be used. Cooking (hereinafter used so as to include heating) can be a batch or continuous process. Preferably, continuously using a rotating cooker which injects steam directly into the krill (or flow containing the krill). Suitably, a horizontal cooker with an internal rotating screw is used. The screw rotates at a low rotation speed of about 1-100 rpm, preferably 2-20 rpm, and more preferably 5-10 rpm, without agitation to avoid the oil emulsification. The krill temperature at entrance to the cooker is at atmospheric temperature and the temperature of the krill at the exit of the cooker is preferably at least about 90° C., more preferably about 90-100° C. and, in some embodiments even more preferably about 93-95° C., or whatever other temperatures not disclosed here but that are necessary to reach complete protein denaturation. The heating of the krill also allows the krill-containing flow, i.e. cooked krill, to be rendered into two phases for later separation: a first solid krill fraction and a first liquid krill fraction. The cooker type is not particularly restricted but as preferred embodiment this cooker must operate without agitation for avoiding the emulsification state. The amount of time the krill is cooked will depend upon several factors including but not limited, the type of cooker used, i.e. batch or continuous, size and temperature, rate of throughout. In some embodiments, the cooking time for batches will be from about 10 to about 20 minutes, while cooking times for continuous processes can have similar time ranges, which corresponds to the approximate residence time of the krill in the continuous cooking device without causing significant emulsification of the krill phospholipids.

In some embodiments, the cooked krill is transported to a separator or decanter or separating decanter 14, preferably a two-phase decanter, using a screw conveyor or a screw pumping, or another transporting system without krill agitation for avoiding oil emulsification. A separator is used to separate the cooked krill into a first solid fraction and a first liquid fraction. Any conventional liquid-solid separator can be used. Suitably, centrifugation is used to accelerate the separation of solid and liquid. Suitably, a centrifuge separator such as decanter centrifuge is used. In some preferred embodiments, a decanter separator is used for separating the solid and liquid. In alternative embodiments, alternative separators such a horizontal decanter separators or similar apparatus can be used.

The cooked krill is preferably passed through a two phase-decanter with high torque about 1-10 kNn, preferably 1.2-5 kNn, more preferably 1.5-3 kNm, even more preferably 1.8-2.5 kNm, and a speed of about 100-10,000 rpm, in some embodiments, preferably 1,000-8,000 rpm, more preferably 2,000-5,000 rpm, even more preferably 3,000-4,600 rpm, for partial de-watering and partial de-fatting, separating it into a decanter solids phase and a decanter liquid phase. In some alternative embodiments, the cooked krill separating is conducted using a decanter operated at speed about 3.000 rpm or higher. The decanter type is not particularly restricted. The moisture content of decanter solid is about 40-80%, preferably, 50-70%, more preferably 55-67%, even more preferably 58-65%.

Table No 1 shows the lipid and moisture content of decanter solids and decanter liquids, (corresponding to the first solid and first liquid krill fractions, respectively) obtained with a krill catch during fatty period using a cooker with indirect and direct steam heating with a screw conveyor at low rotation speed (i.e. about 2-10 rpm). The decanter solid has a lipid content range of 19.8 to 22.4% wt dry base, so the resulting krill meal with 8% of moisture from this solid will have a lipid content of about 18.2% to 20.6%. These results show that the use of a two-phase decanter alone is not enough to achieve a good de-fatting of the cooked krill to obtain more krill oil and krill meal with a fat content lower than 18%. Anyway, the decanter solids obtained from a cooker operating a at a low speed have a lower lipid content and lower non-emulsified lipids than that of decanter solids obtained using processes in which a contheiui cooker is used. The processes of the present invention which involve using a cooker working at a lower speed of rotation advantageously make it possible to avoid emulsification. Moreover, the inventive processes allow the resulting lipids be found more in the liquid fraction instead of the solid fraction. Conversely, when a contherm type cooker which works at a higher speed of rotation (above 500 rpm), emulsification is generated and, as seen in Table 2, below, lipids are retained more in the solid fraction and less in the liquid fraction.

Table 1. Lipid and moisture content of decanter solids and decanter liquids obtained with krill captured during South Antarctic's Krill fatty period using a cooker with indirect and direct steam heating system with screw conveyor operating at a low rotation speed. See also FIG. 2 Flow Chart.

TABLE 1 Krill Tem- Decanter pera- Mois- Total Lipids Lipids Torque ture ture Solids WB DB Krill Material (kNm) (° C.) (%) (%) (%) (%) Decanter Solids 2.0 94 63.1 36.9 7.3 19.8 Decanter Liquid 93.2 6.8 1.6 23.5 Decanter Solids 2.1 93 62.9 37.1 8.1 21.8 Decanter Liquid 92.7 7.3 1.8 24.7 Decanter Solids 2.0 94 61.6 38.4 7.8 20.3 Decanter Liquid 91.3 8.7 1.9 21.8 Decanter Solids 2.0 93 63.0 37.0 8.3 22.4 Decanter Liquid 91.1 8.9 1.9 21.3 WB: wet base DB: dry base

In some embodiments of the present invention, using a cooker calibrated for a low rotation speed of the screw conveyor in a continuous process of preferably about 2-10 rpm. The decanter liquid obtained in such cookers is not emulsified and has higher lipid content compared to the decanter liquid obtained using a conventional contherm cooker.

Following the flow chart of FIG. 4, Table No 2 shows the lipid and moisture content of decanter solids and decanter liquid, wherein a contherm cooker has been used. This equipment is not preferred within the embodiments of this invention, as a large proportion of lipids remain in the decanter solids, and the lipids contained in the decanter liquid are emulsified. Krill oil recovery using this equipment was very low and the resulting krill meal has a high fat content, specifically over 20% when obtained in the krill fatty period.

Table 2. Lipid and moisture content of decanter solids and decanter liquids obtained with krill captured during South Antarctic's Krill fatty period using a contherm cooker system See FIG. 4

TABLE 2 Krill Tem- Decanter pera- Mois- Total Lipids Torque ture ture Solids Lipids DB Krill Material (kNm) (° C.) (%) (%) WB (%) (%) Decanter Solids 2.1 95 63.1 36.9 8.7 23.6 Decanter Liquid 92.7 7.3 1.5 20.5 Decanter Solids 2.0 96 61.9 38.1 9.3 24.4 Decanter Liquid 92.1 7.9 1.7 21.5 Decanter Solids 2.0 94 63.0 37.0 8.4 22.7 Decanter Liquid 92.3 7.7 1.5 19.5 Decanter Solids 2.0 95 63.6 36.4 8.6 23.6 Decanter Liquid 92.8 7.2 1.7 23.6 WB: wet base DB: dry base

While applicants are not bound by theory, it is believed that the combination of the preferred decanting and pressing conditions described herein result in obtaining krill oil compositions having higher levels triglycerides and phospholipids while minimizing emulsion formation by virtue of limited rotational speeds for the device during cooking. Such desirable results are not available when contherm cooking apparatus with their higher rotational speeds are employed. Thus, the low rotational speeds mentioned herein, i.e. about 1-100 rpm, preferably 2-20 rpm, and more preferably 5-10 rpm, are used, without agitation of the krill being cooked, to avoid the oil emulsification.

Turning now to the flow chart of FIG. 2, some details are provided regarding a preferred embodiment in the process of the present invention. After cooking and decanting of the krill, the obtained decanter liquid with neutral lipids and astaxanthin is passed through a centrifuge separator to separate the first liquid fraction (decanter liquid) into oil and water. Any conventional liquid-liquid separator can be used. Suitably, centrifugation is used to accelerate separating the liquids. Suitably, a centrifuge separator is used. After the separator centrifuge a neutral-enriched krill oil and a first stick water (stick water 1 in flow chart) is obtained. The oily fraction is passed again through a purifier separator centrifuge (identified in the flow chart as Polish Centrifuge #1), to separate the krill oil enriched with neutral lipids with astaxanthin and substantially free or without (or no detectable) phospholipids from additional stick water (not shown in the flow chart). The obtained stick water (Stick water 1 in the flow chart) has a low fat content, with a maximum of about 0.3-0.5%, in a non-emulsified form suitable for further processing (concentrate production) and sludge. Stick water can be further evaporated to form a concentrate. See also FIG. 3 for a related process for obtaining krill oil, press cakes, etc.

In some embodiments, there is provided a process for obtaining the neutral lipid-enriched oil which, has an exclusive and separated line including: separator centrifuge, purifier separator centrifuge, pumps, piping, heat exchangers, tanks and packaging station. As such, there is provided an organic solvent-free process for obtaining krill oil-containing compositions having the steps of a) cooking krill in a vessel for a time and at a temperature sufficient to denature the protein content of the krill and cause a first solid krill fraction being partially defatted and de-watered and a first liquid krill fraction to be formed while substantially avoiding emulsification of the first solid and first liquid krill fractions; b) removing the first solid and first liquid krill fractions from the cooker vessel at a temperature of at least about 90° C.; c) separating the first solid fraction and the first liquid fraction; and d) centrifuging the first liquid portion obtained as a result of step b) to obtain an organic solvent free krill oil composition enriched in neutral lipids and stick water. and optionally removing the stick water therefrom.

In a preferred embodiment of the process of the present invention, the decanter solids phase or, the first solid krill fraction, is fed to a press 26 using a screw pump or a screw conveyor or other feeding system wherein agitation does not occur. A press is used to express the second liquid krill fraction 28 from the first solid krill fraction and form a second solid fraction 29. Such presses are conventional and include, for example, single press, twin screw press, double or other presses know to those of ordinary skill. Suitably, squeezing is conducted preferably in a continuous process but may be a batch process. Suitable presses include box presses, platen presses, pot presses, cage press, for a batch process and screw presses, and roller presses for a continuous process. In one aspect of this embodiment, a two screw press is used which squeezes the first solid fraction and separates the squeezed first solid fraction into the second solid fraction and a second liquid fraction. The decanter solids phase is pressed using twin screw press with high pressing force or using another pressing system to release the oil with phospholipids 36 linked to the denatured protein according to the production line for the product of the present invention. As a preferred embodiment, the pressing step is carried out by continuous pressing at a full feeding condition using a 2-10 rpm speed, and more preferably 3-6 rpm and a decanter solids temperature feeding of 90-96° C. and more preferably 93-95° C. The pressing system is not particularly restricted. As described above, the decanter solids phase keeps all of the phospholipids inside of the coagulated protein, then a strong pressing of the decanter solids releases an important high percentage, i.e. up to about 30 to 60%, of the phospholipids to the press liquid. The moisture of this press cake is about 45-55%, preferably 48-53%. Table 3 shows the lipid and moisture content from the press cake of the present invention at different feeding levels of the twin press in the krill fatty period.

TABLE 3 Moisture Dry (%) Solids (%) Lipids WB (%) Lipids DB (%) Press Cake 1 58.1 41.9 8.6 20.5 Press Cake 2 58.6 41.4 8.4 20.3 Press Cake 3 56.9 43.1 8.0 18.5 Press Cake 4 56.7 43.3 8.0 18.5 Press Cake 5 54.6 45.4 7.4 16.3 Press Cake 6 54.5 45.5 7.2 15.8 Press Cake 7 54.3 45.7 5.5 12.0 Press Cake 8 53.9 46.1 5.6 12.1 WB: wet base DB: dry base

Comments

Press Cake samples 1 to 6 were taken when the press was not fully fed with decanter solids

Press Cake samples 7 & 8 were taken when the press was fully fed with decanter solids

Table No 4 shows this press liquid, second liquid fraction, 28, composition in the krill fatty period. According to the process or system disclosed in the present invention, this press liquid has a high fat content in the range of about 3-25%, preferably 5-20% and more preferably 8-17% (wet base), depending on the seasonal lipid content of krill and if it is not in an emulsified form. In the traditional krill meal processing using only a twin-screw press (without prior use of a decanter), the fat content in this press liquid is lower than 0.5-3% (wet base) depending on the lipid content of krill and if it is not in an emulsified form.

TABLE 4 Press Press Composition Liquid 1 Liquid 2 Press Liquid 3 Press Liquid 4 Moisture (%) 78.3 77.7 77.3 80.4 Proteins (%) 5.8 6.1 6.2 5.2 Lipids (%) 16.9 14.0 15.3 13.2

In some embodiments of the present invention, there is also provided a dried complex with oil containing phospholipids with DHA and EPA, proteins and astaxanthin obtained by drying 30 the press liquid “as-is” without centrifuge separation. Such dried complex 32 corresponds to a human-grade krill-related product for being used in many human health applications. See also FIG. 2 in this regard. Drying is done in a conventional manner using conventional equipment. In accordance therewith a process is an organic solvent-free process for obtaining krill oil-containing compositions, having the steps of a) cooking krill in a vessel for a time and at a temperature sufficient to denature the protein content of the krill and cause a first solid krill fraction being partially defatted and de-watered and a first liquid krill fraction to be formed while substantially avoiding emulsification of the first solid and first liquid krill fractions; b) removing the first solid and first liquid krill fractions from the cooker vessel at a temperature of at least about 90° C.; c) separating the first solid fraction and the first liquid fraction; d) squeezing the separated first solid fraction and obtaining a press liquid and a second solid portion therefrom; and e) centrifuging and drying the press liquid obtained as a result of step c) to obtain an organic solvent free dried complex containing krill oil in combination with phospholipids, DHA, EPA, proteins, astaxanthin and neutral lipids.

In some embodiments, a synergic action of the two-phase decanter and further pressing with twin-screw press or another strong pressing system is used to release the phospholipids with DHA and EPA in the oil, mixed with astaxanthin and neutral lipids. The press liquid, at a temperature of about 25-121° C., preferably 50-110° C., more preferably 80-100° C. and even more preferably 90-96° C., is pumped to the separator centrifuge 34, using a screw pump or other feeding system, avoiding agitation, wherein the krill oil with phospholipids with DHA and EPA mixed with astaxanthin and neutral lipids 36 is separated.

The separator that is used to separate the second liquid into oil and water is any conventional liquid-liquid separator. Suitably, centrifugation is used to accelerate the separator of the liquids. Suitably, centrifugation is used to accelerate the separator of the liquids. Suitably, a separator centrifuge is used. The processing speed in the centrifuge separator operates at 4,000-8,000 rpm, more preferably at 4,600-6,800 rpm, with an automatic periodic discharge of solids. In one embodiment of the present invention, the oil separator centrifuge is not particularly restricted, i.e. any centrifuge equipment satisfying the indicated conditions is suitable for being used. This krill oil is once more centrifuged through a purifier separator to clarify it, operating at a processing speed of 5,000-10,000 rpm, more preferably at 6,000-8,200 rpm, with an automatic periodic discharge of solids for later packages. In the embodiments of the present invention, the purifier separator centrifuge is not particularly restricted, i.e. any centrifuge equipment satisfying the indicated conditions is suitable for being used. The stickwater 38 from separator 34 is fed to evaporator 22 to form concentrate 24.

As an additional preferred embodiment, the krill oil with phospholipids with DHA and EPA mixed with astaxanthin and neutral lipids, product of the present invention, is obtained using an exclusive and separated oil process line including: a separator centrifuge, a purifier separator, pumps, piping, heat exchangers, tanks and a packaging station, completely different and separated from the other oil line with neutral lipid from the decanter liquid phase.

Examples 3 and 4, infra, show the characteristics of krill oils obtained in the present invention:

I). the neutral lipid enriched krill oil of the present invention is also useful for health human application having a content of:

-   -   neutral lipids from 50 to 100% w/w, preferably from 60 to 100%         w/w, and more -preferably from 70 to 100% w/w,     -   DHA and EPA content are from 2 to 45% w/w, preferably from 2 to         40% w/w, and more preferably from 5 to 35% w/w,     -   Phospholipids content is lower than 10% w/w, more preferably         lower than 5% w/w, and more preferably lower than 2% w/w,     -   Astaxanthin content is from 200 to 1.500 mg/kg, preferably from         300 to 1200 mg/kg, and more preferably from 400 to 1,000 mg/kg.

II) the phospholipids enriched krill oil of the present invention can be useful for health human applications, having a content of:

-   -   total phospholipids content from 30 to 70% w/w, preferably from         35 to 60% w/w, and more preferably from 35 to 55% w/w,     -   DHA and EPA content is from 10 to 70% w/w, preferably from 15 to         60% w/w, and more preferably from 20 to 55% w/w,     -   Neutral lipids content is from 30 to 70% w/w, preferably from         40% to 65% w/w, and more preferably from 45 to 65% w/w,     -   Astaxanthin content is from 200 to 1.500 mg/kg, preferably from         300 to 1.200 mg/kg, and more preferably from 400 to 1.000 mg/kg.

According to the above declared compositions for both oil products, obtained through the process of the present invention, these oils are suitable for health human applications.

From the above, considering the composition characteristics of this krill oil and the process for obtaining the same, it can be concluded that the process of the present invention provides a krill oil product containing DHA and EPA in the phospholipids fraction with astaxanthin and neutral lipids, which is produced through a different process, which is new and improved, regarding all those krill oils obtained through processes involving solvent extraction and/or using supercritical fluid extraction or through thermal fractionation and centrifugation; additionally, the resulting krill meal, in any Antarctic krill season, can preferably have a maximum fat content of about 15%, a minimum protein content of about 60%, and a maximum moisture content of about 10%.

In some embodiments, krill oils obtained with the procedure disclosed in the present invention can be stabilized by the use of antioxidants and/or preservatives and/or with a nitrogen-barred layer. Moreover, such krill oils can be stored within plastic or metal containers, necessarily suitable for food-grade, pharmaceutical-grade and/or cosmetic/grade applications, in special in stainless steel containers, at room temperature or refrigerated, suitably protected from light.

In some embodiments, the present invention provides uses of krill oils for preparing krill oil compositions for being used as a dietary supplement and/or nutraceutical product. This invention also discloses pharmaceutical compositions comprising an effective amount of krill oil and at least one pharmaceutically acceptable transporter, excipient, stabilizer, diluents and/or adjuvant. In some of the embodiments, said krill oil compositions are suitable as photoprotectors. Said photoprotectors can be formulated as tanning creams and/or tanning oils. In some of the embodiments, said krill oil can be used to enhance cosmetic products. Said cosmetic products are, but no limited to, moisture creams, powder make ups, powder eye shadows, cream eye shadows compact powders and lipsticks, In some of the embodiments, said krill oil compositions can be effectively used for decreasing cholesterol plasma levels, inhibiting platelet adhesion, inhibiting artery plaque formation, preventing hypertension, controlling arthritis symptoms, preventing skin cancer, enhancing transdermal transport, reducing the symptoms of premenstrual symptoms or controlling blood glucose levels in a patient. Furthermore, in some embodiments, nutraceuticals, pharmaceuticals and cosmetics comprising the phospholipids-enriched krill oil are also embraced by the present invention.

EXAMPLES

The present invention will be described in more detail using examples. It should be understood that the present invention is not limited by the following Examples.

Example 1

Process to Obtain Krill Oils and Low Fat Krill Meal of the Present Invention

The description represents an example of the process of the present invention. In FIG. 2 there is depicted a flow diagram of this process. The process clearly does not involve the use of organic solvents and/or supercritical CO₂ fluid.

Antarctic krill was captured during the period of March to May, preferably during the month of May, within krill's fatty period in the Orkney Islands fishing ground, using a pumping catch system, the Krill arriving alive on board a factory vessel and being immediately processed. Table No 5 shows the fresh raw whole krill composition used.

TABLE 5 Compounds Value Moisture (%) 77.2 Proteins (%) 13.7 Lipids (%) 5.3 Ash (%) 3.0 Astaxanthin (mg/Kg) 47

Whole krill was collected into bunkers to drain seawater and transported on belt conveyors to tanks from wherein it was pumped to the cooker, model Stord Disc Cooker RPH-60, using a screw pump. The continuous cooker with screw conveyor used indirect steam heating (of the cooker) and live steam injection (i.e. direct steam contact with the krill) to increase the krill temperature from about 0° C. to 93-95° C. The cooker screw conveyor speed was set at 7-8 rpm.

The cooked krill was then pumped to a Westfalia CD536 two-phase decanter using a screw pump The continuous working cooker with screw conveyor used indirect steam heating and live steam to increase the krill temperature from about 0° C. to 93-95° C. The cooker screw conveyor speed was set at 7-8 rpm. The cooked krill was pumped to a two-phase decanter. The heating is done as a single continuous step to bring the krill up to and thereafter be maintained at the desired temperature >90 C, unlike prior art techniques which have multiple heating and cooling steps.

The two-phase decanter operated at 3,100 rpm and a 2.0 kNm torque was used, resulting in a decanter liquid phase and a decanter solid phase. Table No¹ shows the decanter liquid and decanter solid composition.

The decanter liquid phase was collected into tanks at a temperature of 93-96° C., from wherein it was pumped with a screw pump to a Westfaia SA100 separator centrifuge at a speed set at 4,600 rpm and immediately thereafter passed to a purifier separator at a speed set at 6,100 rpm, for obtaining a krill oil with neutral lipids without phospholipids. Example 3 below shows this neutral lipids oil composition and characteristics.

The decanter solid phase was pressed in a Stord Twin screw press resulting in a press liquid phase and press cake phase. Table No 6 shows the composition obtained from these two phases.

TABLE 6 Compounds Press Liquid (%) Press Cake (%) Moisture 77.7 54.5 Proteins 6.1 32.7 Lipids 14.0 6.0 Ash 0.9 4.6

The press cake was dried until it reached a moisture content lower than 10% using a Atlas Stord Disc Dryer (rotaplate dryer). Example 5 shows the krill meal composition obtained from this test.

The press liquid at a temperature of 93-96° C. was pumped with a screw pump to a specific separator centrifuge at a speed set at 4,600 rpm and immediately thereafter to a specific purifier separator at a speed set 6,100 rpm, to separate the krill oil with phospholipids. Example 4, below, shows the composition characteristics of this krill oil with DHA and EPA linked to the phospholipids fraction obtained from the present experiment.

Example 2

Flow Diagram and Mass Balance of the Process to Obtain Krill Oils and Low Fat Krill Meal of the Present Invention

This example shows an estimate mass balance of a production line for the product of the present invention when raw krill is within the season when fat content is high (estimated at 5%) although not necessarily the highest fat content found in raw South Antarctic Krill and without the addition or recovery of stick water.

The following tables are an estimated mass balance of the process of the present invention without stick water recovery, utilizing raw krill with average-high fat content (around 5% w/w).

TABLE 7 Product MT/Hr Yield (%) Krill Meal (without concentrate) 1.63 16.3 Krill Oil with Phospholipids 0.22 2.2 Krill Oil with Neutral Lipids 0.07 0.7 Total 1.92 19.2

TABLE 8 FRESH RAW WHOLE KRILL Kg/Hr (%) Kg/Hr (%) DS 1,800 18.0 F 500 5.0 Phospholipids 200 40.0 Neutral Lipids 295 59.0 Cholesterol 5 1.0 M 7,700 77.0 Total 10,000 100 500 100

TABLE 9 DECANTER SOLIDS Kg/Hr (%) Kg/Hr (%) DS 1,386 28.7 F 401 8.3 Phospholipids 190 47.4 Neutral Lipids 208 51.9 Cholesterol 3 0.7 M 3,043 63.0 Total 4,830 100 401 100

TABLE 10 PRESS CAKE Kg/Hr (%) Kg/Hr (%) DS 1,248 40.8 F 160 5.2 Phospholipids 104 65.0 Neutral Lipids 54 34.0 Cholesterol 2 1.0 M 1,652 54.0 Total 3,060 100 160 100

TABLE 11 PRESS CAKE + SLUDGE Kg/Hr (%) Kg/Hr (%) DS 1,303 35.6 F 195 5.3 Phospholipids 121 62.4 Neutral Lipids 70 35.9 Cholesterol 3 1.7 M 2,162 59.1 Total 3,660 100 195 100

TABLE 12 KRILL SOLUBLES Kg/Hr (%) DS 0 0.0 F 0 0.0 M 0 0.0 Total 0 0.0

TABLE 13 FEED TO DRIERS RCD Kg/Hr (%) Kg/Hr (%) DS 1,303 35.6 F 195 5.3 Phospholipids 121 62.4 Neutral Lipids 70 35.9 Cholesterol 3 1.7 M 2,162 59.1 Total 3,660 100 195 100

TABLE 14 KRILL MEAL Kg/Hr (%) Kg/Hr (%) DS 1,303 80.0 F 195 12.0 Phospholipids 121 62.4 Neutral Lipids 70 35.9 Cholesterol 3 1.7 M 130 8.0 Total 1,628 100 195 100

TABLE 15 EVAPORATOR Kg/Hr 0

TABLE 16 EVAPORATED DRIERS RCD Kg/Hr 2,032

TABLE 17 DECANTER LIQUID Kg/Hr (%) Kg/Hr (%) DS 414 8.0 F 99 1.9 Phospholipids 10 10.1 Neutral Lipids 87 87.9 Cholesterol 2 2.0 M 4,657 90.1 Total 5,170 100 99 100

TABLE 18 OIL WITH NEUTRAL LIPIDS Kg/Hr (%) Kg/Hr (%) Oil 69 100 Phospholipids 0 0 Neutral Lipids 68 98.6 Cholesterol 1 1.4 Total 69 100 69 100

TABLE 19 PRESS LIQUID Kg/Hr (%) Kg/Hr (%) DS 139 7.8 F 241 13.6 Phospholipids 86 35.7 Neutral Lipids 153 63.7 Cholesterol 1 0.6 M 1,391 78.6 Total 1,770 100 241 100

TABLE 20 OIL WITH PHOSPHOLIPIDS Kg/Hr (%) Kg/Hr (%) Oil 220 100 Phospholipids 79 35.7 Neutral Lipids 140 63.7 Cholesterol 1 0.3 Total 220 100 219 100

TABLE 21 SLUDGE 1 Kg/Hr (%) Kg/Hr (%) DS 41 10.5 F 18 4.5 Phospholipids 10 56.1 Neutral Lipids 7 38.3 Cholesterol 1 5.6 M 336 85.0 Total 395 100 18 100

TABLE 22 SLUDGE 2 Kg/Hr (%) Kg/Hr (%) DS 14 6.8 F 17 8.2 Phospholipids 7 44.2 Neutral Lipids 9 51.7 Cholesterol 1 4.2 M 174 85.0 Total 205 100 17 100

TABLE 23 STICK WATER 1 Kg/Hr (%) Kg/Hr (%) DS 372 7.9 F 12 0.3 Phospholipids 0 0 Neutral Lipids 12 100 Cholesterol 0 0 M 4,322 91.8 Total 4,706 100 12 100

TABLE 24 STICK WATER 2 Kg/Hr (%) Kg/Hr (%) DS 125 9.3 F 4 0.3 Phospholipids 0 0 Neutral Lipids 5 100 Cholesterol 0 0 M 1,216 90.4 Total 1,345 100 5 100 DS = Dry Solids Content F = Fat Content M = Moisture Content MT = Metric Ton

The data from the above tables show that the inventive process allows more fat to found in the liquid portion and thus allows easier recovery. See Table 19 in particular with regard to the level of phospholipids in the press liquid and Table 17 with regard to the level of triglycerides in the decanted liquid.

The following tables shows an estimate mass balance of a production line for the product of the present invention when raw krill is within the season when fat content is high (estimated at 5%) although not necessarily the highest fat content found in raw South Antarctic Krill and with the addition or recovery of stick water.

TABLE 25 Production MT/Hr Yield (%) Krill Meal (with concentrate) 2.19 21.9 Krill Oil with Phospholipids 0.22 2.2 Krill Oil with Neutral Lipids 0.07 0.7 Total 2.47 24.7

TABLE 26 FRESH RAW WHOLE KRILL Kg/Hr (%) Kg/Hr (%) DS 1,800 18.0 F 500 5.0 Phospholipids 200 40.0 Neutral Lipids 295 59.0 Cholesterol 5 1.0 M 7,700 77.0 Total 10,000 100 500 100

TABLE 27 DECANTER SOLIDS Kg/Hr (%) Kg/Hr (%) DS 1,386 28.7 F 401 8.3 Phospholipids 190 47.4 Neutral Lipids 208 51.9 Cholesterol 3 0.7 M 3,043 63.0 Total 4,830 100 401 100

TABLE 28 PRESS CAKE Kg/Hr (%) Kg/Hr (%) DS 1,248 40.8 F 160 5.2 Phospholipids 104 65.0 Neutral Lipids 54 34.0 Cholesterol 2 1.0 M 1,652 54.0 Total 3,060 100 160 100

TABLE 29 PRESS CAKE + SLUDGE Kg/Hr (%) Kg/Hr (%) DS 1,303 35.6 F 195 5.3 Phospholipids 121 62.4 Neutral Lipids 70 36.1 Cholesterol 3 1.5 M 2,162 59.1 Total 3,660 100 195 100

TABLE 30 KRILL SOLUBLES Kg/Hr (%) Kg/Hr (%) DS 497 48.5 F 16 1.5 Phospholipids 0 0 Neutral Lipids 16 100 Cholesterol 0 0 M 513 50.0 Total 1,026 100 16 100

TABLE 31 FEED TO DRIERS RCD Kg/Hr (%) Kg/Hr (%) DS 1,800 38.4 F 211 4.5 Phospholipids 121 57.6 Neutral Lipids 86 41.0 Cholesterol 3 1.4 M 2,675 57.1 Total 4,686 100 211 100

TABLE 32 KRILL MEAL Kg/Hr (%) Kg/Hr (%) DS 1,800 82.4 F 211 9.6 Phospholipids 121 57.6 Neutral Lipids 86 41.0 Cholesterol 3 1.4 M 175 8.0 Total 2,185 100 211 100

TABLE 33 Kg/Hr EVAPORATOR 5,025

TABLE 34 Kg/Hr EVAPORATED DRIERS RCD 2,500

TABLE 35 DECANTER LIQUID Kg/Hr (%) Kg/Hr (%) DS 414 8.0 F 99 1.9 Phospholipids 10 10.1 Neutral Lipids 87 87.9 Cholesterol 2 2.0 M 4,657 90.1 Total 5,170 100 99 100

TABLE 36 OIL WITH NEUTRAL LIPIDS Kg/Hr (%) Kg/Hr (%) Oil 69 100 Phospholipids 0 0 Neutral Lipids 68 98.6 Cholesterol 1 1.4 Total 69 100 69 100

TABLE 37 PRESS LIQUID Kg/Hr (%) Kg/Hr (%) DS 139 7.8 F 241 13.6 Phospholipids 86 35.7 Neutral Lipids 153 63.7 Cholesterol 1 0.6 M 1,391 78.6 Total 1,770 100 241 100

TABLE 38 OIL WITH PHOSPHOLIPIDS Kg/Hr (%) Kg/Hr (%) Oil 220 100 Phospholipids 79 35.7 Neutral Lipids 140 63.7 Cholesterol 1 0.5 Total 220 100 220 100

TABLE 39 SLUDGE 1 Kg/Hr (%) Kg/Hr (%) DS 41 10.5 F 18 4.5 Phospholipids 10 56.1 Neutral Lipids 7 38.3 Cholesterol 1 5.6 M 336 85.0 Total 395 100 18 100

TABLE 40 SLUDGE 2 Kg/Hr (%) Kg/Hr (%) DS 14 6.8 F 17 8.2 Phospholipids 7 44.2 Neutral Lipids 9 53.5 Cholesterol 0 2.4 M 174 85.0 Total 205 100 17 100

TABLE 41 STICK WATER 1 Kg/Hr (%) Kg/Hr (%) DS 372 7.9 F 12 0.3 Phospholipids 0 0 Neutral Lipids 12 100 Cholesterol 0 0 M 4,322 91.8 Total 4,706 100 12 100

TABLE 42 STICK WATER 2 Kg/Hr (%) Kg/Hr (%) DS 125 9.3 F 4 0.3 Phospholipids 0 0 Neutral Lipids 4 100 Cholesterol 0 0 M 1,216 90.4 Total 1,345 100 4 100

Example 3

Composition of the Neutral Lipid Enriched Krill Oil of the Present Invention

TABLE 43 % w/w Neutral Lipids Triglycerides 84.6 Diglycerides 4.9 Free Fatty Acids ND Monoglycerides ND Total 89.5 Phospholipids ND <0.5 alpha-Tocopherol 0.7 FAME (Fatty Acid Fatty acid Analysis Methyl Ester) % w/w Total Sample  8:0 0.0  9:0 0.0 10:0 0.0 11:0 0.0 12:0 0.3 13:0 0.0 14:0 16.3 15:0 0.5 16:0 18.2 17:0 0.3 18:0 1.5 19:0 1.0 20:0 0.0 22:0 0.1 23:0 0.0 24:0 0.0 Saturated Total 38.3 11:1 0.0 13:1 0.0 14:1 0.2 16:1 9.5 17:1 1.2 18:1 cis 14.6 18:1 trans 6.4 20:1 1.2 22:1 0.1 24:1 0.0 Monounsaturated Total 33.2 18:2 1.3 18:3 (6, 9, 12) 0.2 18:3 (9, 12, 15) 0.6 20:2 0.0 20:3 (8, 11, 14) 0.0 20:4 0.0 20:3 (5, 8, 11) 0.2 20:5 3.3 22:2 0.2 22:3 0.0 22:4 0.0 22:5 N3 0.1 22:6 1.1 Polyunsaturated Total 7.1 Total All 78.6 ND = Not Detected

Example 4

Composition of the Phospholipids Enriched Krill Oil of the Present Invention

TABLE 44 Composition (% total lipid)* Composition Preferred Lipid Class (% total lipid)* ranges Sterol esters 1.2   1-1.5 Triacylglycerols 45.6 40-50 Free fatty acids 3.4 2-4 Cholesterol/sterols 4.3 3-5 Diacylglycerols** 7.7  5-10 Monoacylglycerols 0.9 0.2-1.5 Total neutral lipids 63.1 Phosphatidylcholine 25.2 23-29 Phosphatidylethanolamine 7.3 6-9 Phosphatidylserine 1.5 0.7-3.7 Phosphatidylinositol 1.3 0.9-2.2 Phosphatidylglycerol + cardiolipin 1.0 0.6-1.4 Lysophosphatidylcholine 0.6   0-1.2 Total polar lipids 36.9 Values are means of duplicate analyses *as determined by HPTLC/densitometry. **contains pigments.

The low levels of lysophosphatidylcholine (LPC) found in compositions made in accordance with the processes of the invention are noteworthy. Lysophosphatidylcholine (LPC) is the resulting compound of the decomposition of Phosphatidylcholine (PC) throughout the entire extraction process and/or raw frozen krill storage normally through an extended period of time. The lower the content of LPC is the result of processing krill very fresh and limited decomposition. Results of a process that works with raw material that has not gone through different deterioration steps. This low LPC content is a very distinctive parameter of our invention as we are working with extremely fresh raw material, processed on board from fresh raw krill in the same fishing ground where it is captured. Other processes extract the oil on shore, from a raw material that has been previously pre-processed on board (but later re-processed on shore) Prior art methods result in PL oils with LPC above 5%.

Fatty Acid Composition of Total Lipid

TABLE 45 % total fatty acids Preferred ranges +/− 20% for each value More Preferred ranges +/− 10% for each value 14:0 12.55 br 15:0 0.28 15:0 0.48 br 16:0 0.13 16:0 21.67 br 17:0 0.43 17:0 0.10 18:0 1.47 20:0 0.29 Total saturated 37.39 16:1n-9 0.38 16:1n-7 6.81 16:1 isomer 0.60 17:1n-8 0.29 18:1n-9 12.21 18:1n-7 7.43 20:1n-11 0.07 20:1n-9 0.93 20:1n-7 0.42 22:1n-11 0.53 22:1n-9 0.14 24:1n.9 0.21 Total monounsaturated 30.02 18:2n-6 1.74 18:3n-6 0.17 20:2n-6 0.08 20:3n-6 0.10 20.4n-6 0.33 Total n-6 PUFA 2.43 (Polyunsaturated Fatty Acids) 18:3n-3 0.98 18:4n-3 3.24 20:3n-3 0.10 20:4n-3 0.34 20:5n-3 15.20 22:5n-3 0.40 22:6n-3 7.55 Total n-3 PUFA 27.81 16:2 0.69 16:3 0.27 16:4 1.38 Total 16C PUFA 2.35 TOTAL PUFA 32.59 Total 100.00

Values are means of duplicate analyses

Limit of quantification (LOQ) for fatty acid analysis=0.06%

Fatty Acid Composition of Phosphatidylcholine/Lysophosphatidylcholine

TABLE 46 % total fatty acids Preferred ranges +/− 20% for each value More Preferred ranges +/− 10% for each value 14:0 2.79 br 15:0 0.10 15:0 0.37 br 16:0 0.12 16:0 27.17 br 17:0 0.47 17:0 0.15 18:0 1.04 20:0 0.22 Total saturated 32.44 16:1n-9 0.15 16:1n-7 1.73 16:1 isomer 0.28 17:1n-8 0.14 18:1n-9 4.46 18:1n-7 5.14 20:1n-11 <LOQ 20:1n-9 0.60 20:1n-7 0.27 22:1n-11 1.12 22:1n-9 0.50 24:1n.9 0.46 Total monounsaturated 14.87 18:2n-6 1.55 18:3n-6 0.27 20:2n-6 <LOQ 20:3n-6 0.10 20.4n-6 0.59 Total n-6 PUFA 2.50 18:3n-3 1.05 18:4n-3 1.98 20:3n-3 0.14 20:4n-3 0.47 20:5n-3 31.50 22:5n-3 0.63 22:6n-3 14.09 Total n-3 PUFA 49.87 16:2 0.10 16:3 0.12 16:4 0.11 Total 16C PUFA 0.33 TOTAL PUFA 52.70 Total 100.00

Values are means of duplicate analyses

Limit of quantification (LOQ) for fatty acid analysis=0.06%

Fatty Acid Composition of Phosphatidylethanolamine

TABLE 47 % total fatty acids Preferred ranges +/− 20% for each value More Preferred ranges +/− 10% for each value 14:0 0.56 br 15:0 0.07 15:0 0.20 br 16:0 0.17 16:0 14.65 br 17:0 0.39 17:0 0.83 18:0 1.24 20:0 <LOQ Total saturated 18.11 16:1n-9 0.25 16:1n-7 0.61 16:1 isomer 0.27 17:1n-8 <LOQ 18:1n-9 3.92 18:1n-7 14.03 20:1n-11 <LOQ 20:1n-9 0.48 20:1n-7 0.10 22:1n-11 <LOQ 22:1n-9 <LOQ 24:1n.9 0.08 Total monounsaturated 19.79 18:2n-6 0.95 18:3n-6 0.49 20:2n-6 0.15 20:3n-6 0.15 20.4n-6 1.35 Total n-6 PUFA 3.10 18:3n-3 0.38 18:4n-3 0.25 20:3n-3 0.14 20:4n-3 0.42 20:5n-3 23.35 22:5n-3 0.75 22:6n-3 33.12 Total n-3 PUFA 58.43 16:2 0.14 16:3 0.17 16:4 0.27 Total 16C PUFA 0.58 TOTAL PUFA 62.10 Total 100.00

Values are means of duplicate analyses

Limit of quantification (LOQ) for fatty acid analysis=0.06%

Fatty Acid Composition of Triacylglycerol

TABLE 48 % total fatty acids Preferred ranges +/− 20% for each value More Preferred ranges +/− 10% for each value 14:0 20.52 br 15:0 0.42 15:0 0.61 br 16:0 0.15 16:0 21.85 br 17:0 0.48 17:0 0.09 18:0 1.67 20:0 0.31 Total saturated 46.11 16:1n-9 0.21 16:1n-7 9.90 16:1 isomer 0.72 17:1n-8 0.36 18:1n-9 16.76 18:1n-7 8.26 20:1n-11 0.08 20:1n-9 1.14 20:1n-7 0.55 22:1n-11 0.24 22:1n-9 0.11 24:1n.9 <LOQ Total monounsaturated 38.32 18:2n-6 1.79 18:3n-6 0.22 20:2n-6 <LOQ 20:3n-6 <LOQ 20.4n-6 0.08 Total n-6 PUFA 2.09 18:3n-3 0.86 18:4n-3 3.40 20:3n-3 0.07 20:4n-3 0.17 20:5n-3 4.05 22:5n-3 0.20 22:6n-3 1.49 Total n-3 PUFA 10.22 16:2 1.00 16:3 0.37 16:4 1.88 Total 16C PUFA 3.25 TOTAL PUFA 15.57 Total 100.00

Values are means of duplicate analyses

Limit of quantification (LOQ) for fatty acid analysis=0.06%

Lipid class composition of lipid samples and fatty acid compositions of individual lipids were determined by high performance thin-layer chromatography (HPTLC) and quantitation using a scanning densitometry according to Henderson and Tocher (Henderson, R. J. and Tocher, D. R. (1992) Thin-layer chromatography. In Lipid Analysis: A Practical Approach (Hamilton, R. J., and Hamilton, S., eds.) pp. 65-111, Oxford University Press, Oxford).

Example 5

Other Characteristics of the Phospholipid Enriched Krill Oil of the Present Invention

TABLE 49 Composition Preferred ranges +/− 20% for each value More Preferred ranges +/− 10% for each value Vitamin E 13.80 Composition (*) in mg/Kg Peroxide Value (*) 0.0 in Meq/Kg Iodine Value (IV) (**) 150.37 (g/100 g) (*) Calculated in Duplicate (**) Calculated by the modification of Ham et al. (J.A.O.C.S. 75, 1445-1446(1998)) of the AOCS recommended practice Cd 1c-85.

Example 6

Heavy Metals Content for the Phospholipid Enriched Krill Oil of the Present Invention

TABLE 50 Present Neptune Invention Bioressouces 999 (Triple Nine) Heavy Metal %/w %/w %/w Antimony ppm <0.02 <0.03 <0.02 Arsenic ppm <0.05 2.31 4.49 Bismuth ppm <0.02 0.17 <0.02 Cadmium ppm <0.02 <0.03 0.07 Copper ppm <0.04 0.21 13.1 Lead ppm <0.02 0.09 0.27 Mercury ppm <0.02 <0.03 <0.04 Molybdenum ppm <0.02 <0.03 0.06 Silver ppm <0.02 <0.03 0.45 Tin ppm <0.02 <0.03 0.05 Total ppm <0.05 2.78 18.49

Example 7

Composition of the Low Fat Krill Meal of the Present Invent with Krill Captured during South Antarctic's Krill Fatty Period

TABLE 51 Compounds Value Moisture (%) 8.0 Proteins (%) 66.1 Lipids (%) 12.1 Ash (%) 9.3 Astaxanthin (mg/Kg) 119

Example 8

Dietary Supplement (Nutraceutical/Dietary) Based on Krill Oil

It is described a food rich in fat material for complementing essential fatty acids. Such food was formulated in cookie form elaborated using krill oil, krill meal or krill dried complex.

In a bowl 400 g Quaker oats was mixed with 100 g of flour, 500 g of sugar, 1 egg, 150 mL of krill oil of this invention and 10 mL of vanilla extract. Once completely homogenized, the cookies were molded weighing each 25 g, and were baked at 160° C. for 15 min. as longer cooking times destroys the astaxanthin

The amount of cookies used in the diets will depend on the amount of essential fatty acids such as 18:2 and 18:3 required, and the necessary calories.

Example 9

Photoprotector

The krill oil prepared as described in the examples 3 and 4 can be used for the preparation of tanning creams and tanning oils for solar protection.

A) Tanning Creams.

In this example are described two tanning creams, one with solar protection factor 5 (SPF5) and one with solar protection factor 20 (SPF20).

TABLE 52 Composition per 100 g of Tanning Cream Component SPF 5 SPF 20 Krill Oil 5.00 g 5.00 g Carbopol 940 0.25 g 0.25 g Cetilic alcohol 0.50 g 0.50 g Methyl paraben 0.15 g 0.15 g Estearic acid III pr. 2.00 g 2.00 g Propyl paraben 0.15 g 0.15 g Panalen 5.00 g 5.00 g EDTA disodium 0.10 g 0.10 g Triethanolamine 0.50 g 0.50 g Propylenglycol 2.50 g 2.50 g Octylmethoxycinamate — 7.50 g Benzophenone 3 — 3.00 g Titanium dioxide — 0.50 g Imidazolidinilurea 0.30 g 0.30 g Glyceril monoestearate 1.00 g 1.00 g Natural essence c.s. c.s. Isopropyl miristate 2.00 g 2.00 g Water c.s. c.s. B) Tanning Oil:

The following description corresponds to tanning oil that contains krill oil as the unique sun-blocking agent.

TABLE 53 Compound Composition per 100 g of Tanning Oil Cosmetic liquid Vaseline 80.0 g Hydrogenated polybutenes  5.0 g Krill oil 14.7 g Essence  0.3 g

Example 10

Cosmetic Products Based on Krill Oil

Since krill oil has several biological activities, such as being a pigmentant, antioxidant capacity, EPA and essential fatty acids content, it is possible to design cosmetic products.

A) Moisturizing Cream.

TABLE 54 Composition per 100 g Compound of Moisturizing Cream Estearic acid 1.0 g Anionic glyceril monoestearate A.E. 0.7 g Neutral glyceril monoesteararte 0.5 g Hydrogenated polybutenes 3.2 g Krill oil 10.0 g  Propylenglycol 2.0 g Isopropyl miristate 1.5 g Carboxyvinyl polymer 0.3 g Propylparabene 0.1 g Methylparabene 0.1 g Essence 0.3 g EDTA disodium 0.2 g Triethanolamine 99% 1.2 g Demineralized water 78.9 g  B) Powder Makeup.

In this example it is described a base formula for the elaboration of powder makeup that contains 10% (w/w) krill oil.

TABLE 55 Composition per Compound 100 g of Makeup Talcum powder 52.56 g Krill oil 10.00 g Mica:Titanuim dioxide (2:1) 26.00 g Magnesium estearate 3.50 g Isopropyl myristate 1.60 g Oleic alcohol 2.60 g Octylpalmitate 3.40 g Methylparabene 0.17 g Propylparabene 0.17 C) Powder Eye Shadow.

In this example it is described a base formula for the elaboration of a powder eye-shadow that contains 10% (w/w) krill oil of this invention.

TABLE 56 Compound Composition per 100 g of Eye Shadow Talcum powder 51.46 g  Krill oil 10.00 g  Mica:Titanium dioxide (3:1) 27.00 g  Magnesium estearate 3.50 g Isopropyl miristate 1.60 g Oleic alcohol 2.60 g Octylpalmitate 3.50 g Methylparabene 0.17 g Propylparabene 0.17 g D) Cream Eye Shadow

In this example, a base formula for the elaboration of a cream eye shadow that contains 5.7% (w/w) krill oil of this invention is described.

TABLE 57 Composition per Compound 100 g of Eye Shadow Talcum powder 4.0 g Estearic acid 9.5 g Isoestearic acid 1.9 g Krill Oil 5.7 g Titanium dioxide 1.9 g Aluminum and magnesium silicate 3.6 g Propylenglycol 16.1 g  Triethanolamine 11.0 g  Methylparaben 0.2 g Propylparabene 0.2 g Deionized water 45.9 g  E) Compact Powder.

In this example, a base formula for the elaboration of a compact powder that contains 10% (w/w) krill oil of the present invention is described.

TABLE 58 Composition per Compound 100 g of Compact Powder Kaolin 32.0 g  Krill Oil 10.0 g  Mica:Titanium dioxide (2:1) 1.6 g Magnesium estearate 4.1 g Octylpalmitate 2.5 g Propylparabene 0.5 g Talcum powder 49.3 g  F) Lipstick

In this example, a base formula for a lipstick that contains 3% (w/w) krill oil is described.

TABLE 59 Compound Composition per 100 g of Lipstick Castor Oil 42.04 g  Krill Oil 3.00 g Oleic alcohol 2.60 g Lanolin 25.00 g  Sorbitan monoestearate 1.30 g Ozokerite 4.50 g Carnauba wax 5.20 g Beeswax 5.60 g Estearic acid 4.90 g Candle wax 5.60 g Methylparabene 0.13 g Propylparaben 0.13 g

Example 11

Pharmaceutical Products Based on Krill Oil

Krill and/or marine oil has been shown to decrease cholesterol in vivo. It also inhibits platelet adhesion and plaque formation and reduces vascular endothelial inflammation in a patient. It can offer hypertension prophylaxis. It prevents oxidation of low-density lipoprotein. It may have an inhibitory effect on the secretion of VLDL due to increased intracellular degradation of apo B-100. It also offers a post-myocardial infarction prophylaxis because of its ability to decrease CIII apolipoprotein B, to decrease CIII non-apolipoprotein B lipoproteins and to increase antithrombin III levels. Krill and/or marine oil is suitable for prophylactic usage against cardiovascular disease in human where cardiovascular disease relates to coronary artery disease, hyperlipidemia, hypertension, ischemic disease (relating to angina, myocardial infarction, cerebral ischemia, shock without clinical or laboratory evidence of ischemia, arrhythmia).

A pharmaceutical composition of krill oil of this invention comprises capsules containing 1 mL of krill oil described in examples 3 and 4. A pharmaceutical composition of krill dried complex of this invention comprises capsules containing 1 to 5 g of krill dried complex. 

What we claim is:
 1. An organic solvent-free process for producing krill oil, comprising: a) cooking krill in a cooker vessel for a time and at a temperature sufficient to denature the protein content of the krill and cause a first solid krill fraction and a first liquid krill fraction to be thrilled while substantially avoiding emulsification of the first solid and first liquid krill fractions; b) removing the first solid and first liquid krill fractions from the cooker vessel at a temperature of at least about 90° C.; c) separating the first solid fraction and the first liquid fraction; and d) obtaining krill oil from either the first liquid fraction or the first solid fraction, said separating and said obtaining steps being carried out without the use of organic solvents.
 2. A process according to claim 1, wherein the krill is harvested and cooked at sea.
 3. The process of claim 1, wherein the cooking is carried out using indirect steam heating and live steam injection.
 4. The process of claim 1, wherein the first solid and first liquid krill fractions being removed from the cooker vessel have a temperature of from about 90 to about 100° C.
 5. The process of claim 2, wherein the cooking is conducted within about 4 hours of harvesting the krill and without freezing the krill before cooking.
 6. The process of claim 1, wherein the cooking is conducted in a horizontal cooker vessel equipped with an internal screw conveyor with indirect steam heating which rotates to move the krill from an entrance of the cooker vessel to an exit of the cooker vessel the screw conveyor rotating at about 1 to about 100 rpm; and live steam being injected into the cooker vessel to cook the krill.
 7. The process of claim 1, wherein the separating is conducted using a decanter operated at speed about 3,000 rpm or higher.
 8. The process of claim 7, wherein the separating is conducted using decanter operated at a torque of about 1 to about 10 KN-m.
 9. The process of claim 1, wherein the obtaining krill oil from the first solid fraction comprises squeezing the first solid fraction to obtain a second solid fraction. and a second liquid fraction; and separating the second liquid fraction therefrom to obtain krill oil enriched with phospholipids and water.
 10. The process of claim 9, wherein the squeezing is conducted in a two screw press, wherein the screws thereof are rotated at about 2 to 10 rpm and the first solid fraction has a temperature of about 90° to about 100° C. when fed to the two screw press.
 11. The process of claim 9, wherein the separating of the second liquid fraction is conducted in a centrifuge separator.
 12. The process of claim 9, wherein the separating of the second liquid fraction is conducted by centrifuging the second liquid fraction to remove water and obtain the krill oil enriched in phospholipids.
 13. The process of claim 1, wherein the obtaining of the krill oil from the first liquid fraction, comprises separating the first liquid fraction into a krill oil enriched in neutral lipids and water.
 14. The process of claim 10, wherein the separating of the first liquid fraction is conducted in a centrifuge separator.
 15. An organic solvent-free process for obtaining krill oil-containing compositions, comprising: a) cooking krill in a vessel for a time and at a temperature sufficient to denature the protein content of the krill and cause a first solid krill fraction being partially defatted and de-watered and a first liquid krill fraction to be formed while substantially avoiding emulsification of the first solid and first liquid krill fractions; b) removing the first solid and first liquid krill fractions from the cooker vessel at a temperature of at least about 90° C.; c) separating the first solid fraction and the first liquid fraction; d) squeezing the separated first solid fraction and obtaining a press liquid and a second solid portion therefrom: and e) centrifuging the press liquid obtained as a result of step d) to obtain an organic solvent free krill oil composition enriched in phospholipids.
 16. An organic solvent-free process for obtaining krill oil-containing compositions, comprising: a) cooking krill in a vessel for a time and at a temperature sufficient to denature the protein content of the krill and cause a first solid krill fraction being partially defatted and de-watered and a first liquid krill fraction to be foamed while substantially avoiding emulsification of the first solid and first liquid krill fractions; b) removing the first solid and first liquid krill fractions from the cooker vessel at a temperature of at least about 90° C.; c) separating the first solid fraction and the first liquid fraction; d) centrifuging the first liquid portion obtained as a result of step c) to obtain an organic solvent free krill oil composition enriched in neutral lipids and stick water and optionally removing the stick water therefrom.
 17. An organic solvent-free process for obtaining krill oil-containing compositions, comprising: a) cooking krill in a vessel for a time and at a temperature sufficient to denature the protein content of the krill and cause a first solid krill fraction being partially defatted and de-watered and a first liquid krill fraction to be formed while substantially avoiding emulsification of the first solid and first liquid krill fractions; b) removing the first solid and first liquid krill fractions from the cooker vessel at a temperature of at least about 90° C; c) separating the first solid fraction and the first liquid fraction; and d) squeezing the separated first solid fraction and obtaining a press liquid and a second solid portion therefrom; and e) and drying the press liquid obtained as a result of step d) to obtain an organic solvent free dried complex containing krill oil in combination with phospholipids, MHA, EPA, proteins, astaxanthin and neutral lipids.
 18. An organic solvent-free process for producing fish or algae extracts, comprising: a) cooking fish or algae or portions thereof in a cooker vessel for a time and at a temperature sufficient to denature the protein content of the fish or algae and cause a first solid fraction and a first liquid fraction to be formed while substantially avoiding emulsification of the first solid and first liquid fractions; b) removing the first solid and first liquid fractions from the cooker vessel at a temperature of at least about 90° C.; c) separating the first solid fraction and the first liquid fraction; and d) obtaining the fish or algae extract from either the first liquid fraction or the first solid fraction, said separating and said obtaining steps being carried out without the use of organic solvents. 